The present invention relates to a gene detection chip and detection device capable of detecting and analyzing gene base sequences as well as gene abnormalities such as genetic DNA single base substitution SNPs (single nucleotide polymorphisms: mutations of human genetic code), multiple base substitutions, point mutations, and genetic defects.
Methods in which probe DNA is immobilized on an electrode, the probe DNA is hybridized with sample DNA, and the resulting hybrid is electrochemically detected have been proposed as a means of detecting the base sequence of gene DNA (see Japanese Patent Laid-open No. 9-288080 and Proceedings of the 57th Meeting of the Japan Society of Analytical Chemistry, pp. 137-138, 1996) These methods allow such hybrids to be detected with high sensitivity.
There exist, however, enormous numbers of such single base genetic substitution SNPs, genetic mutations, and the like, so at least 2,000,000 single base genetic substitution SNPs must be identified in order, for example, to map such single base substitution SNPs with a density (resolution) of 15 kB in humans. Genetic point mutations related to existing disorders also exist in large numbers. A means that can be used to comprehensively analyze such single base substitutions or point mutations.
The present invention, which was perfected in view of this situation, provides a gene detection chip and detection device that allow large amounts of genes to be detected (that is, processed with a high throughput) and allow detection and analysis procedures to be carried out with high sensitivity.
The gene detection device of the present invention comprises a plurality of pins that constitute measurement electrodes, and a common electrode that constitutes a counter electrode for these measurement electrodes. At least portions of the surfaces of the abovementioned pins are coated with a resin. Here, a so-called detection chip is included in the meaning of the term xe2x80x9cdetection devicexe2x80x9d. Furthermore, a device constructed by attaching a detection chip to a measuring device is also included in the meaning of the term xe2x80x9cdetection devicexe2x80x9d.
In the above arrangement, the pins may be configured such that the resin is applied only to part of the surface on which the Au film is formed. Covering only part of the outermost surface of the pins with resin makes it possible to immobilize a gene solely on the uncoated, exposed portions and allows the exposed surface area to be kept constant and the amount of immobilized probe to be controlled, producing detection results that have higher sensitivity. In addition, an arrangement in which the lateral surface of each pin is covered with resin and the gene is immobilized solely on the exposed portion (located at the end opposite from the base end of the pin fixedly supported on the supporting member) is preferred because the exposed surface area can be readily determined based on the thickness of the pin electrodes. The coating resin should be PEEK (polyether ether ketone), a fluororesin or other thermoplastic resin, or an epoxy resin because of considerations related to chemical resistance.
In the above, the abovementioned pins may contact the surface of a supporting member or may be implanted in the surface of a supporting member, and the lateral surfaces of these pins and the portions of the surface of the abovementioned supporting member that are not contacted by these pins or in which these pins are not implanted may be respectively coated with a resin. In this case, the abovementioned resin is preferably a fluororesin. In particular, a copolymer of tetrafluoroethylene and hexafluoropropylene is especially desirable.
In the above, the device may comprise a plate-form member which consists of a resin and which has a plurality of pin holes into which the abovementioned plurality of pins are respectively inserted, and portions of the surfaces of the abovementioned pins may be coated by this plate-form member.
According to the present invention, a plurality of pins can be secured easily and stably because the pins are inserted into and held in place inside the pin holes with the aid of a plate-form member provided with a plurality of pin holes at positions that match the pin positions.
Each pin can be tightly fitted into the corresponding pin hole when inserted thereinto as a result of the fact that the pin hole diameter is the same as or slightly smaller than the outside diameter of the pin. In other words, each pin can be held airtightly by the plate-form member, preventing the solutions used in the detection process from penetrating through the contact areas between the pins and pin holes.
The present invention provides a gene detection device characterized in that the diameters of the pin holes taper off in the direction in which the pins are inserted, and the pins are held in place in the narrowest sections of the pin holes.
According to the present invention, the pin hole diameters vary in tapered fashion, and the pins are inserted from the side with the large diameter and are held in place in the section with the minimum hole diameter, thus facilitating positioning when the pins are inserted into the pin holes and making it easier to conduct operations in which the tabular member is mounted on the pins. Another feature is that because the minimum hole diameter is the same as or slightly less than the pin diameter, the pins are held airtightly by the plate-form member when the pins are inserted into the pin holes, and the solutions used in the detection process can be prevented from penetrating through the contact areas between the pins and pin holes.
It is desirable that the abovementioned pins contact the surface of the supporting member or be implanted in the surface of the supporting member, and that the abovementioned plate-form member adhere tightly to the surface of the abovementioned supporting member.
According to the present invention, the pins are held in place while the plate-form member is tightly bonded to the supporting member, making it possible to prevent the solutions used in the detection process from penetrating through the joint between the plate-form member and the supporting member. The supporting member may also be a circuit substrate whose interior contains electric circuitry.
The plate-form member should preferably contain a thermoplastic resin as the principal component thereof. Specifically, PTFE (polytetrafluoroethylene) and other fluororesins can be cited as examples of suitable materials, as can PEEK (polyether ether ketone). Using a plate-form member composed of a thermoplastic resin makes it possible to provide a gene detection chip that has excellent heat resistance and is highly resistant to the chemicals (alkalis, acids, and the like) used for detection pretreatments. Such enhanced chemical and heat resistance makes this material suitable for processes in which a detection cycle is followed by the removal of sample DNA and the re-hybridization of another sample DNA to allow the gene detection chip to be reused, or for processes in which a probe gene is removed and another probe gene is re-immobilized on the pins to allow the gene detection chip to be reused.
It is also possible to fabricate the present detection device by a process in which the heat-shrinkage properties of a thermoplastic resin (for example, PEEK) are utilized to form a tabular member whose pin holes have somewhat enlarged diameters, pins are inserted into the pin holes, the plate-form member is heat-treated to induce heat shrinkage, and the diameters of the pin holes are reduced to cause the pin holes to constrict the pins. Adopting this arrangement facilitates positioning when pins are inserted into the pin holes because the diameters of the pin holes are greater than the outside diameters of the pins, makes it easier to mount the plate-form member on the pins, and allows the pins to be held airtightly in a state in which the pins and the pin holes are tightly joined with each other.
A particularly preferred feature of the present invention is that the plate-form member be composed of PTFE (polytetrafluoroethylene). PTFE is highly flexible and can be used to airtightly hold the pins in the pin holes. In addition, the high flexibility of PTFE makes this resin suitable for creating a tight bond between the plate-form member and the ceramic supporting member.
A particularly preferred feature of the present invention is that the pins consists of an Au (gold) film on the surface of an alloy primarily containing Fe (iron), Ni (nickel), and Co (cobalt).
Using pins in which an Au film is formed on the surface of an Fexe2x80x94Nixe2x80x94Co alloy is preferred from the standpoint of mass production. Such an allow is preferred because it has a coefficient of linear expansion of between 5.0xc3x9710xe2x88x926/xc2x0 C. and 9.0xc3x9710xe2x88x926/xc2x0 C., and can easily conform to the expansion of a ceramic material. An alloy having such a coefficient of linear expansion may, for example, be obtained by adding 15-20 wt % of an Ni component, 25-30 wt % of a Co component, and 50-60 wt % of an Fe component. The alloy may also contain no more than 1.0 wt % of impurities.
The thickness of the Au film should preferably be kept 20 xcexcm or less to achieve good coverage of the substrate and to prevent cost overruns due to the formation of an excessively thick film. The Au film should preferably be an Au plating formed by conventional electroplating. It is also possible to perform Ni plating as a surface preparation treatment that precedes Au plating, and to subsequently form an Au plating thereon.
Because the present invention features the above-described pins, genes can be electrochemically detected with high sensitivity.
Using an array of pins allows a plurality of genes to be analyzed at the same time. As used herein, the term xe2x80x9carrayxe2x80x9d refers to a large number of pins arranged such that they extend parallel to each other from a specific surface.
The gene detection device of the present invention comprises a supporting member for fixedly supporting one end of each pin, with the supporting member primarily composed of a ceramic. The supporting member contains a ceramic as its principal component, and hence has excellent chemical resistance and strength. The ceramic should preferably be alumina (Al2O3), silicon carbide (SiC), silicon nitride (Si3N4), zirconia (ZrO2), beryllia, or the like because of considerations related to chemical resistance. Alumina is particularly suitable. The ceramic should preferably have a coefficient of linear expansion of between 6.0xc3x9710xe2x88x926/xc2x0 C. and 11.0xc3x9710xe2x88x926/xc2x0 C. to facilitate joining with the pins. The supporting member should preferably contain at least 90 wt % of the ceramic.
In the above arrangement, a plurality of PCR products having identical or different gene sequences, such oligonucleotides, mRNA, cDNA, PNA (peptidic nucleic acid), and LNA (Locked Nucleic Acids from Proligo LLC), may be immobilized on the pins.
The above-described gene detection device may, for example, be used to detect gene base sequences, single base substitution SNPs, multiple base substitutions, point mutations, translocations, defects, amplifications, and triplet repeats. The presence or absence of genes related to monogenic disorders (such as muscular dystrophy, hemophilia, and phenyl ketonuria) and multifactorial genetic diseases (such as diabetes, cancer, hypertension, myocardial infarction, and obesity) can be diagnosed, or premorbid genes can be diagnosed by genetic screening based on the use of the inventive detection chip, which can thus be employed as a diagnostic material for selecting an appropriate treatment or drug.
Furthermore, the present invention provides a gene detection chip which comprises a plurality of pins that constitute measurement electrodes, and in which at least portions of the surfaces of the abovementioned pins are coated with a resin. This gene detection chip may also be a chip which has a common electrode that constitutes a counter electrode for the abovementioned plurality of pins. Specifically, the detection chip of the present invention may be a chip which has both pin electrodes and a common electrode, or may be a chip which has only pin electrodes with no common electrode, and in which measurements are performed by attaching the detection chip to a solution tank that is equipped with a common electrode.
The abovementioned pins may contact the surface of the supporting member or be implanted in the surface of the supporting member, and the lateral surfaces of the abovementioned pins and the portions of the surface of the supporting member that are not contacted by these pins or in which these pins are not implanted may be respectively coated with a resin.
Furthermore, the device may comprise a plate-form member which consists of a resin and which has a plurality of pin holes into which the abovementioned plurality of pins are respectively inserted, and portions of the surfaces of the abovementioned pins may be coated by this plate-form member.
Furthermore, the abovementioned pins may be pins in which an Au film is formed on the surface of an alloy whose main components are Fe, Ni and Co. The present invention also provides the above-described gene detection chip and a measurement device capable of accepting or releasing this detection chip.
The present invention provides a gene detection chip which comprises a plurality of pins that constitute measurement electrodes, and a common electrode that constitutes a counter electrode for these measurement electrodes, and in which the abovementioned pins are formed by forming an Au film on the surface of an alloy whose main components are Fe, Ni and Co.
It is desirable that the abovementioned pins contact the surface of the supporting member or be implanted in the surface of the supporting member, and that the abovementioned supporting member be constructed with a ceramic as the main component of this supporting member.
It is desirable that the abovementioned pins contact the surface of the supporting member or be implanted in the surface of the supporting member, and that the abovementioned supporting member be constructed with alumina as the main component of this supporting member.
Large amounts of genes can thus be concurrently detected through simple operations with extremely high sensitivity by employing the above-described chip to allow electrochemically active molecules to bind following hybridization or to perform hybridization in the presence of electrochemically active molecules.